The present invention, in some embodiments thereof, relates to novel chemical conjugates and to uses thereof in therapy and diagnosis and, more particularly, but not exclusively, to novel conjugates of polymers having attached thereto an angiogenesis targeting moiety and an anti-angiogenesis agent and to uses thereof in monitoring and treating medical conditions associated with angiogenesis.
Angiogenesis is a biological process that involves the sprouting of new blood vessels from pre-existing ones and plays a crucial role in disease development and progression. Angiogenesis is a complex process in which endothelial cells serve as a building block for blood vessel expansion. This process involves an extensive interplay among cells, growth factors, and extracellular membrane (ECM) components. It is regulated through a fine balance between pro-angiogenic and anti-angiogenic molecules.
Pathological angiogenesis has been demonstrated in several diseases, including atherosclerosis, cancer, hypertension, rheumatoid arthritis, diabetes and diabetes related complications such as diabetic retinopathy. Tumor growth and metastasis are particularly dependent on the degree of angiogenesis. Tumor angiogenesis is the proliferation of a network of blood vessels that penetrate into cancerous tumors in order to supply nutrients and oxygen and remove waste products, thus leading to tumor growth. Tumor angiogenesis involves hormonal stimulation and activation of oncogenes, expression of angiogenic growth factors, extravasation of plasma protein, deposition of a provisional ECM, degradation of ECM, and migration, proliferation and elongation of endothelial capillaries.
Inhibition of further vascular expansion has therefore been the focus of active research for cancer therapy. Many drugs have been developed, which target different steps in this multi-step tumor angiogenesis process. However, most of these drugs were shown to be cytostatic rather than cytotoxic and thus do not cause a substantial reduction of tumor volume during the first stage of treatment.
There are currently eight approved anti-cancer therapies with recognized antiangiogenic properties. These agents, which interrupt critical cell signaling pathways involved in tumor angiogenesis and growth, can be divided into two primary categories: (1) monoclonal antibodies directed against specific proangiogenic factors and/or their receptors; (Avastin, Erbitux, Vectibix, Herceptin) and (2) small molecule tyrosine kinase inhibitors (TKIs) of multiple proangiogenic growth factor receptors (Tarceva, Nexavar, Sutent, Iressa). Inhibitors of mTOR (mammalian target of rapamycin) represent a third, smaller category of antiangiogenic therapies with one currently approved agent (Torisel). In addition, at least two other approved angiogenic agents may indirectly inhibit angiogenesis through mechanisms that are not completely understood (Velcade, Thalidomide/Celgene)
The first FDA-approved angiogenesis inhibitor, Bevacizumab (Avastin, Genentech) a monoclonal antibody to vascular endothelial growth factor (VEGF), has recently been approved for metastatic colon cancer treatment in conjunction with standard conventional chemotherapy.
The largest class of drugs that block angiogenesis are the multi-targeted tyrosine kinase inhibitors (TKIs) that target the VEGF receptor (VEGFR). These drugs such as sunitinib (Sutent, Pfizer), Sorafenib (Nexavar, Bayer/Onyx Pharmaceuticals) and Erlotinib (Tarceva, Gennentech/OSI/Roche) have the advantages of hitting multiple targets, convenient oral administration, and cost effectiveness. While these drugs exhibit promising efficacy, their use is limited by their lack of target specificity, which leads to unexpected toxicity [Cabebe et al. Curr Treat Options Oncol 2007; 8:15-27].
Tumor endothelial cells are drug sensitive for long time periods and may be treated with cytotoxic agents in an “anti-angiogenic dosing schedule”. This dosing schedule involves the administration of chemotherapy in low doses, well below the maximum tolerated dose (MTD), in close intervals for extended periods of time (“metronomic dosing”). As a result, acute toxicity should be avoided and the drugs may be administered during longer periods, eventually converting cancer to a chronic manageable disease.
The microtubule-interfering agent Paclitaxel (PTX) is a clinically well-established and highly-effective anti-neoplastic drug used as a monotherapy and in combination therapy mainly for the treatment of prostate, breast, ovarian, and non-small cell lung cancers and it is the drug of choice for the treatment of metastatic breast cancer. It has also shown anti-angiogenic and pro-apoptotic properties [Oldham et al. 2000 Int. J Oncol. 16:125-132]. However, due to the hydrophobic nature of the drug, solubilizing agents such as Cremophor EL or ethanol are required for its administration. PTX causes severe adverse side effects such as neutropenia, neuropathies, and when solubilized in Cremophor EL causes hypersensitivity reactions. In addition, only a small amount of the drug localizes in the tumor and the drug is substrate to efflux pumps in particular p-glycoprotein, resulting in multiple drug resistance.
Water-soluble copolymers such as hydroxypropyl methacrylate (HPMA) and PGA are biocompatible, non-immunogenic and non-toxic carriers that enable specific delivery into tumor tissue (Satchi-Fainaro et al. Nat Med 2004; 10: 255-261). These macromolecules do not diffuse through normal blood vessels but rather accumulate selectively in the tumor site because of the enhanced permeability and retention (EPR) effect. This phenomenon of passive diffusion through the hyperpermeable neovasculature and localization in the tumor interstitium is observed in many solid tumors for macromolecular agents and lipids.
Conjugation of anti-cancer drugs to copolymers, such as HPMA or PGA, has been suggested so as to restrict the passage through the blood brain barrier and to prolong the circulating half-life of the drugs, hence inhibiting the growth of tumor endothelial and epithelial cells by exposing the cells to the conjugated drugs in the circulation for a longer time compared to the free drugs.
U.S. Pat. No. 6,884,817 teaches compositions comprising a chemotherapeutic and/or anti-angiogenic drug, conjugated to a water-soluble polyamino acid or soluble metal chelator. The taught compositions provide water soluble taxoids which overcome the drawbacks associated with the insolubility of the drugs themselves, and further improve the delivery of the drugs to tumor tissue and affect a controlled release of the conjugated drug. According to the teachings of in U.S. Pat. No. 6,884,817, an exemplary such conjugate of the anti-cancer drug paclitaxel and polyglutamate, exhibited superior antitumor activity together with a reduced level of toxicity, as compared with the anti-tumor agent paclitaxel alone.
The conjugate paclitaxel-polyglutamate OPAXIO™ (paclitaxel poliglumex, CT-2103) (Formerly known as XYOTAX™) showed promising results in phase III trials and is currently being evaluated for marketing approval.
U.S. patent application Ser. No. 12/117,678 having publication No. 2008/0279778 also teaches polyglutamate polymers conjugated to a plurality of drugs for use in drug targeting, stabilizing and imaging applications. A HPMA copolymer conjugate of paclitaxel has also been described by Meerum Terwogt et al. [Anticancer drugs 2001; 12:315-323]. This conjugate was aimed at improving drug solubility and providing controlled release of paclitaxel. In this conjugate, the paclitaxel is linked to the HPMA copolymer through an ester bond, and is hence released from the polymer by non-tissue specific hydrolytic or enzymatic (esterases) degradation of the ester bond, thereby inducing the commonly observed toxicities of paclitaxel.
WO 03/086382 teaches conjugates of water-soluble polymers and the anti-angiogenesis agent TNP-470, and their use as anti-tumor agents, in particular their use as carriers of TNP-470 into tumor vessels, and their effect on the neurotoxicity of TNP-470. According to the teachings of WO 03/086382, an exemplary such conjugate, HPMA-(TNP-470) conjugate (caplostatin), exhibited superior antitumor activity together with a reduced level of toxicity, as compared with TNP-470 alone.
WO 03/086178 teaches a method for decreasing or inhibiting disorders associated with vascular hyperpermeability by the administration of an effective amount of an anti-angiogenesis compound or a compound capable of increasing cell-cell contacts by stabilizing tight junction's complexes and increasing contact with the basement membrane. The compounds taught by WO 03/086178 are endostatin, thrombospondin, angiostatin, tumastatin, arrestin, recombinant EPO, and polymer conjugated TNP-470. According to the teachings of WO 03/086178, an exemplary such conjugate, HPMA-(TNP-470) inhibited vascular endothelial growth factor (VEGF)-induced vessel hyperpermeability and inhibited endothelial cell mediated angiogenesis both in vitro and in vivo.
Integrins are a class of receptors involved in the mechanism of cell adhesion. Alterations in the function of these receptors are responsible for the occurrence of a number of pathologic manifestations, such as, for example, defective embryogenesis, blood coagulation, osteoporosis, acute renal failure, retinopathy and cancer, particularly metastasis. Since the 1980s it is well recognized that integrins play a key role in cell matrix interactions and hence in angiogenesis.
The integrins are heterodimeric transmembrane glycoproteins that compose a diverse family of 19α and eight β subunits. An integrin with a well-characterized involvement in angiogenesis and tumor invasiveness is αvβ3. The αvβ3 integrin is a molecular marker that differentiates newly formed capillaries from their mature counterparts. This integrin is expressed in various malignant tumors. Inhibition of the αvβ3 mediated cell matrix interaction leads to apoptosis of activated endothelial cells and to disruption of blood vessel formation. In contrast, αvβ3 is not strongly expressed on quiescent endothelial cells, thus treatment with αvβ3 antagonists does not affect pre-existing blood vessels [Brooks et al. Science 1994; 264:569-571].
αvβ3 integrins therefore play an important role in adhesion, motility, growth and differentiation of endothelial cells. αvβ3 integrins are known to bind the RGD sequence (Arg-Gly-Asp; SEQ ID NO:1), which constitutes the recognition domain of different proteins, such as laminin, fibronectin and vitronectin. The RGD sequence represent the minimal amino acid domain, in several extracellular matrix proteins, which has been demonstrated to be the binding site of the transmembrane integrins proteins family [Bazzoni et al. 1999, Current Opinion in Cell Biology; (11) pp. 573-581]. Indeed it was shown that replacement of even a single amino acid of this short sequence results in loss of binding activity to integrins. Integrins mediates the attachment of endothelial cells to submatrix proteins that form the basement membrane of the capillary. Although all endothelial cells use the integrin to anchor to the extraluminal submatrix, the αvβ3 integrins are found on the luminal surface of the endothelial cell only during angiogenesis. Thus, agents that target this integrin actually target endothelial cells involved in angiogenesis.
The αvβ3 integrin is overexpressed on proliferating endothelial cells such as those present in growing tumors, as well as on some tumor cells of various origins. Expression of endothelial αvβ3 integrin receptor in aggressive breast carcinomas and glioblastomas is a marker of poor prognosis.
It has been demonstrated that RGD-containing peptides, either isolated from phage peptides library or biochemically synthesized, were able to compete with extracellular matrix proteins on binding to integrins [Haubner et al. 1997, Angew. Chem. Int. Ed. Engl.; (36) pp. 1374-1389]. Tumor-induced angiogenesis can be targeted in vivo by antagonizing the αvβ3 integrin with small peptides containing the RGD amino acid sequence.
It has been further found that the substrate specificity of RGD-containing peptides results from the different conformations of the RGD sequence in different matrix proteins. For example, the bis-cyclic peptide E-[c(RGDfK)2] (SEQ ID NO:2) is a ligand-based vascular-targeting agent that binds to integrin αvβ3.
Encoded by a growth factor-inducible immediate-early gene, Cyr61 (also known as CCN1) is a cysteine-rich matricellular protein that supports cell adhesion and induces adhesion signaling. Furthermore, Cyr61 stimulates endothelial cell migration and enhances growth factor induced DNA synthesis in culture and therefore induces angiogenesis in vivo. Mechanistically, Cyr61 acts as a non-RGD-containing ligand of integrin receptors. Functional blockade of αvβ3, a Cyr61 integrin receptor, is specifically cytotoxic towards Cyr61-overexpressing breast cancer cells and a specific αvβ3-RGD peptidomimetic agent prevents αvβ3 from binding to its ligand, Cyr61.
Cyr61 plays a key role in both the maintenance and the enhancement of a malignant phenotype in breast cancer. Cyr61 is overexpressed in about 30% of invasive breast carcinomas, whereas Cyr61 expression levels in normal breast tissues are negligible. It has been recently shown that Cyr61 overexpression can render human breast cancer cells highly resistant to Paclitaxel. Pharmacological interference with the Cyr61/αvβ3 interaction fully restores Paclitaxel efficacy in Cyr61 overexpressors, thus implying that a previously unrecognized Cyr61/αvβ3-driven cellular signaling actively modulates breast cancer cell growth and chemosensitivity.
Chen et al. reported [J. Med. Chem. 2005; 48:1098-1106] the synthesis and antitumor activity of paclitaxel (PTX) conjugated with a bis-cyclic RGD (E[RGDyK]2) (SEQ ID NO:3) in a metastatic breast cancer cell line.
Mitra et al. report [Journal of Controled Release 2006; 28: 175-183] the biodistribution and tumor targeting properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based conjugates of mono-(RGDfK) (SEQ ID NO:4) and doubly cyclized (RGD4C; SEQ ID NO:5) αvβ3 binding peptides.
WO 2006/012355 teaches an anti-angiogenic polymer conjugate (APC) for treatment of solid tumors comprising a chemical moiety targeting cell-surface proteins of endothelial cells at an angiogenic site. The chemical moiety taught in the application may be a ligand such as RGD4C (SEQ ID NO:5) or RGDfK (SEQ ID NO:4) for a cell-surface receptor, such as, for example, an integrin. The polymer conjugate taught by WO 2006/012355 may further comprise at least one side chain comprising a chelator capable of chelating a pharmaceutically acceptable radioactive label. The scintigraphic images and biodistribution of an exemplary such conjugate, HPMA copolymer-RGD4C-99mTc conjugate (SEQ ID NO: 6), indicated specific in vivo tumor targeting as well as prolonged retention of the conjugate at the tumor site. Treatment of SCID mice bearing DU145 human prostate carcinoma xenografts with another exemplary conjugate, HPMA copolymer-RGD4C comprising the beta particle emitter 90Y (SEQ ID NO:7), resulted in significant decrease of tumor volume as compared to the control (also reported by Mitra et al. in [Nuclear Medicine and Biology 2006; 33:43-52])
Wan et al. [2003 Proc. Intl Symp. Control. Rel. Bioact. Mater. Vol 30: 491-492] teach targeting endothelial cells using HPMA copolymer-doxorubicin-RGD conjugates (SEQ ID NO:8).